Hindawi Publishing Corporation

Mediators of Inammation
Volume 2014, Article ID 263786, 12 pages
http://dx.doi.org/10.1155/2014/263786

Research Article
Niacin Inhibits Vascular Inflammation via Downregulating
Nuclear Transcription Factor-B Signaling Pathway
Yanhong Si,1,2 Ying Zhang,1 Jilong Zhao,3 Shoudong Guo,1 Lei Zhai,1 Shutong Yao,1,2
Hui Sang,1,2 Nana Yang,1 Guohua Song,1 Jue Gu,4 and Shucun Qin1
1

Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Taishan Medical University,
Shandong 271000, China
2
College of Basic Medical Sciences, Taishan Medical University, Shandong 271000, China
3
Shandong Agricultural University, Shandong 271000, China
4
Department of Geriatrics, PLA Navy General Hospital, Beijing 100048, China
Correspondence should be addressed to Jue Gu; gujue0705@163.com and Shucun Qin; shucunqin@hotmail.com
Received 9 January 2014; Revised 1 April 2014; Accepted 16 April 2014; Published 27 May 2014
Academic Editor: Clara Di Filippo
Copyright 2014 Yanhong Si et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The study aimed to investigate the effect of niacin on vascular inflammatory lesions in vivo and in vitro as well as its lipid-regulating
mechanism. In vivo study revealed that niacin downregulated the levels of inflammatory factors (IL-6 and TNF-) in plasma,
suppressed protein expression of CD68 and NF-B p65 in arterial wall, and attenuated oxidative stress in guinea pigs that have
been fed high fat diet. In vitro study further confirmed that niacin decreased the secretion of IL-6 and TNF- and inhibited NF-B
p65 and notch1 protein expression in oxLDL-stimulated HUVECs and THP-1 macrophages. Moreover, niacin attenuated oxLDLinduced apoptosis of HUVECs as well. In addition, niacin significantly lessened lipid deposition in arterial wall, increased HDLC and apoA levels and decreased TG and non-HDL-C levels in plasma, and upregulated the mRNA amount of cholesterol 7hydroxylase A1 in liver of guinea pigs. These data suggest for the first time that niacin inhibits vascular inflammation in vivo and in
vitro via downregulating NF-B signaling pathway. Furthermore, niacin also modulates plasma lipid by upregulating the expression
of factors involved in the process of reverse cholesterol transport.

1. Introduction
Niacin (nicotinic acid, vitamin B3) is a water-soluble vitamin.
In 1955, Altschul et al. reported for the first time that pharmacological doses of niacin can lower plasma cholesterol level
in normal persons as well as hypercholesterolemic patients
[1]. Several subsequent clinical studies established the usage
of niacin as a broad-spectrum lipid-regulating medication.
Niacin alone or in combination can slow or reverse the progression of atherosclerosis (AS) and reduce cardiovascular
event rates and total mortality in patients with hypercholesterolemia and atherosclerotic cardiovascular disease [2, 3].
These effects are generally attributed to favorable actions
on the lipoprotein profile, which includes LDL-C reduction
and HDL-C elevation. However, it is not clear whether the
beneficial effects of niacin on cardiovascular disease can be
completely explained by alterations of plasma lipids.

Recent studies indicated niacin also has the antiinflammatory and antioxidant properties. In human aortic
endothelial cells in vitro, niacin significantly suppressed the
adhesion and accumulation of monocytes/macrophages and
inhibited angiotensin II-induced reactive oxygen species
(ROS) production and LDL oxidation [4], but it is unknown
whether niacin has the same effects or not in vivo. In addition, Kuvin et al. confirmed that extended-release niaspan
not only had a beneficial lipid-regulating effect but also
decreased C-reactive protein (CRP) by 15% in patients with
stable coronary artery disease [5]. CRP is one of the most
important inflammatory markers. Therefore, we hypothesize
that niacin has vascular anti-inflammatory and potentially
vascular-protective property independent of its effect on lipid
regulation.
Guinea pigs have been successfully used to study the
damage of hyperlipidemia [6]. Like human, guinea pig is

2
one of the few species that carry the majority of cholesterol
in LDL [7]. It is possible to induce the initial stages of
AS quickly by feeding them high fat diet [8]. In addition,
significant atherogenic inflammation with an increase of
aortic cytokines has been documented in guinea pigs [7]. In
the study, we select guinea pigs as animal model to study the
protective effect of niacin on vascular inflammatory lesions
induced by high fat diet in vivo. Meanwhile, to further
confirm the direct anti-inflammatory property of niacin, its
effect on oxLDL-induced inflammation of endothelial cells
and macrophages is also explored.

2. Materials and Methods

2.1. Animal and Experimental Design. Thirty-two male England short-hair guinea pigs (260310 g; 5 months old) were
bought from the Animal Laboratory Center of Taibang Biological Products Co., Shandong, China. All experiments were
approved by the Laboratory Animals Ethical Committee of
Taishan Medical University and abided by national guidelines
for the care and use of animals. All guinea pigs were randomly
divided into 4 groups: regular chow diet group (CD), high
fat diet group (HFD, 10% lard + 10% yolk power + 0.30%
cholesterol + 79.7% grass), HFD with niacin group (HFD-N,
HFD + 100 mg/kg/d niacin), and HFD with simvastatin group
(HFD-S, HFD + 20 mg/kg/d simvastatin). Each group is
assigned 8 guinea pigs. The drugs (niacin or simvastatin) were
fed by oral gavage once daily for 8 weeks.
2.2. Cell Culture. Human umbilical vein endothelial cells
(HUVECs, EA.hy926) and THP-1 (human monocyte) were
all purchased from Shanghai Institute of Biochemistry. They
were maintained in RPMI 1640 medium (HyClone, China)
supplemented with 10% fetal bovine serum (HyClone, China)
in a 5% CO2 incubator at 37 C. To induce monocytes
differentiation into macrophages, THP-1 cells were cultured
with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma)
for 24 hours, as described previously [9].
2.3. Immunohistochemistry Examination. Tissue sections
(5 m) from formalin-fixed, paraffin-embedded specimens
were stained with specific antibodies against NF-B p65 and
CD68 proteins (Zhongshan Biotechnology Co., Ltd., Beijing,
China), respectively. The sections were developed with 3,3 diaminobenzidine (Vector Laboratories) and counterstained
with Mayers hematoxylin (Saturatedard Allen). Images were
captured using microscope (Olympus). All quantifications
were examined by calculating the percentage of integrate
optical density (IOD) of the antigen positive staining to the
entire cross-sectional vessel wall by Image-Pro Plus software.
2.4. Analysis of Lipid Deposition in the Arterial Wall. The
proximal aorta attached to the heart was applied to prepare
cryosections. Cryosections (8 m) were cut, gathered, and
stained with oil red O. The quantification of stained lipids was
examined by calculating the percentage of the positive area to
the total cross-sectional vessel wall area by using Image-Pro
Plus software 4.5 (Media Cybernetics). The percentage was
calculated from five sections from an animal.

Mediators of Inflammation
2.5. Measurement of Inflammatory Factors. Plasma concentrations of IL-6, TNF-, and CRP, which can be secreted during the process of inflammation, were determined by ELISA
kit (American R&D) in accordance with the manufacturers
instructions.
ELISA kits were also used for the measurement of TNF and IL-6 protein levels in the medium of HUVECs and
THP-1 macrophages. Cells were incubated in the absence
or presence of niacin (0.251 mM) for 24 h in serum-free
media and then stimulated by 150 g/mL ox-LDL (Beijing
Xiesheng Biotechnology Co. Ltd., China) for 6 h. TNF- and
IL-6 protein levels were assessed, respectively. Results were
calculated as ng/L and expressed as a percentage of those
obtained with blanks.
2.6. Measurement of Oxidative Stress Reaction in Plasma.
Plasma level of malondialdehyde (MDA), a marker for oxidative stress, was determined by a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS)
in accordance with the manufacturers instructions (Nanjing
Jiancheng Bioengineering Institute, China).
2.7. Plasma Lipid Analysis. After being treated by niacin or
simvastatin for 8 weeks, blood was collected by cardiac puncture from guinea pigs without dietary exposure for 12 hours.
Concentrations of plasma total cholesterol (TC), triglyceride
(TG), and HDL cholesterol (HDL-C) were determined by
enzymatic methods (BioSino, Beijing, China). Non-HDL-C
was calculated as TC minus HDL-C.
2.8. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Apolipoproteins in HDL. Lipoprotein isolation was carried out by sequential ultracentrifugation in a LE-80 K ultracentrifuge (Beckman Coulter, Inc.
Brea, CA, USA) as described before. Separation was done
according to the following density fractionation: <
1.019 g/mL for VLDL and IDL; d 1.0191.09 g/mL for LDL;
and d 1.091.24 g/mL for HDL [10]. The isolated specimens
were dialyzed in 150 mmol/L NaCl and 0.3 mmol/L EDTA
at 4 C. HDL containing equal amounts of cholesterol was
loaded on a 15% sodium dodecyl sulfate (SDS) polyacrylamide gradient gel and apolipoprotein samples were stained
with coomassie brilliant blue as described by Jiang et al. [11].
Meanwhile, the marker (Invitrogen, LC5800) was put in lane
1 and HDL from human was put in the last lane for contrast.
Stained gels were scanned and analyzed by Quantity One
(Bio-Rad, Hercules, CA, USA) software program.
2.9. Analysis of Cell Apoptosis by Flow Cytometry. Annexin
V-FITC/PI double-staining assay was applied to measure
apoptosis according to the manufacturers instructions. After
HUVECs were stimulated by ox-LDL for 24 h, cells were
centrifuged, washed twice with PBS, resuspended in 500 uL
binding buffer, and incubated with 5 uL fluorescein isothiocyanate (FITC)-labeled Annexin V and 5 uL propidium
iodide (PI) for 10 minutes at room temperature in the dark.
The scatter parameters of cells were analyzed by FAC Scan
flow cytometer and Cell Quest analysis software (BectonDickinson, CA, USA). Four cell populations were discerned

Mediators of Inflammation
according to the following status: live cells in the lower-left
quadrant (low-PI and FITC signals), early apoptotic cells in
the lower-right quadrant (low-PI and high-FITC signals), late
apoptotic or necrotic cells in the upper-right quadrant (highPI and high-FITC signals), and necrotic cells in the upper-left
quadrant (high-PI and low-FITC signals).
2.10. Western Blot. The entire proteins from fresh aortic
walls or treated cells were extracted using RIPA lysis buffer.
Then the nuclear protein fraction was prepared by a nuclear
protein extraction kit (BestBio, China) in accordance with
the manufacturers instructions. Equal amounts of protein
were subjected to 8% to 15% SDS-PAGE and transferred
onto PVDF membranes by electroblotting. After blocking
in Tris-buffered saline (TBS) containing 0.1% Tween 20
and 10% nonfat dry milk for 2 h at room temperature, the
membranes were incubated with primary antibodies for
3 h at room temperature or overnight at 4 C. After being
washed four times with TBS containing 0.1% Tween 20, the
membranes were incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h at room temperature.
Immunoblots were revealed by ECL reaction and visualized
using a high-performance chemiluminescence film. The IOD
value of immunoreactive bands was measured by Image-Pro
Plus software and normalized by house-keeping protein (actin or histone H3).
2.11. Quantitative Real-Time PCR. RT-PCR assay was applied
to detect the expression of LDL receptor (LDL-R), scavenger receptor Class B Type 1 (SR-B1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and cholesterol 7hydroxylase A1 (CYP7A1) in liver. The first two are receptors of plasma cholesterol and the rest are key enzymes
of cholesterol metabolism. Primer Design 4.1 Software
was used to design the following primers: -actin: forward primer: 5 -TTACTACTTTGCTGCGTTACACC-3 ,
reverse primer: 5 -CATGCCAATCTCATCTCGTTT-3
(length of 78 bp); LDL-R: forward primer: 5 -GACGTGTCCCAGAGGAAGAT-3 , reverse primer: 5 -CGAGTCGGTCCAGTAGATGTT-3 (length of 144 bp); SR-B1: forward
primer: 5 -TCTCCCACCCGCATTTCT-3 , reverse primer:
5 -CGCATACTGCACGTAGCACA-3 (length of 317 bp);
CYP7A1: forward primer: 5 -CAGTATGCTGCTGTTTATG-3 , reverse primer: 5 -GTTCTCGGTGGTGTTTCC-3
(length of 335 bp); HMGR: forward primer: 5 -TGATAGCACCAGCAGATT-3 , reverse primer: 5 -TATAAAGGTTGCGTCCAG-3 (length of 68 bp). The primers were synthesized by TaKaRa. Total liver RNA was separated by TRIZOL Reagent (Invitrogen). cDNA synthesis was performed
using MuLV Reverse Transcriptase (Applied Biosystems).
Real-time PCR was performed using a SYBR-green PCR
master mix kit (TianGen Biotech). The data was analyzed by
using Rotor-gene Q software ver.1.7 (Qiagen). Relative mRNA
levels were calculated by the 2Ct method and normalized
against -actin. Each experiment was repeated three times.
2.12. Statistical Analysis. Results are shown as the mean SD
for at least three independent experiments. Statistical analysis
was performed using one-way analysis of variance (ANOVA)

3
Table 1: Effect of niacin and simvastatin on plasma inflammatory
markers (CRP, IL-6, and TNF-) of guinea pig fed high fat diet.
Group
CD
HFD
HFD-N
HFD-S

CRP (ng/L)
3169.9 219.7
3211.3 153.8
3023.1 180.6
2955.7 257.8

IL-6 (ng/L)
258.8 25.2
265.8 24.2
215.2 38.5
236.7 21.6

TNF- (ng/L)
130.8 9.2
143.0 15.7
117.9 17.9
114.4 19.3

The contents of CRP, IL-6, and TNF- in guinea pig plasma were measured
by ELISA method after 8 weeks treatment. Data are presented as mean SD
( = 8). < 0.05; < 0.01 versus HFD group.

followed by Student-Newmann-Keuls multiple comparison

tests with the SPSS 13.0 software for Windows. values less
than 0.05 were considered statistically significant.

3. Results
At the beginning of the experiment, 32 guinea pigs were
divided into 4 groups randomly and mean initial body weight
was 302.27 23.67 g. All guinea pigs survived for 8 weeks
in the experiment and mean final body weight was 384.89
26.18 g. No significant differences were observed among these
groups for both the initial and final mean body weights.
3.1. Niacin Attenuated the Systemic and Aortic Inflammation
in Guinea Pigs Fed High Fat Diet
3.1.1. Niacin Significantly Downregulated IL-6 and TNF-
Levels in Plasma of Guinea Pigs Fed High Fat Diet. Inflammatory process within the vessel wall can lead to vascular
dysfunction and cause cardiovascular disease. In this process,
inflammatory factors play a key role. In this study, the levels
of three major inflammatory factors (CRP, IL-6, and TNF) in plasma were measured. Compared with CD group,
high fat diet for 8 weeks lightly increased the levels of
CRP, IL-6, and TNF- in plasma, but the increase was not
statistically significant ( > 0.05). Compared with HFD
group, niacin decreased IL-6 level by 19% and decreased
TNF- level by 18%, whereas its effect on CRP had no
statistical difference. Simvastatin decreased the levels of three
inflammatory markers in plasma compared with HFD group
(Table 1).
3.1.2. Niacin Suppressed Protein Expression of CD68 and NFB p65 in the Arterial Wall of Guinea Pigs Fed High Fat
Diet. The invasion of monocyte into arterial intima and its
differentiation into resident macrophages may contribute to
arterial inflammation in experimental atherosclerosis and
hypertension. In the study, the immunohistochemical examination of CD68 protein in the vessel wall was done to
mark monocytes/macrophages. Densitometric quantitative
immunohistochemistry imaging revealed that, compared
with CD group, the positive staining of CD68 was significantly increased in HFD group ( < 0.01); compared with
HFD group, niacin and simvastatin significantly decreased
the densitometric value of positive staining (Figures 1(a)

Mediators of Inflammation

CD

CD

HFD

HFD-N

HFD-S

HFD

HFD-N

HFD-S

(a)

(c)
60

##

40

30

20

10

CD

HFD

HFD-N

HFD-S

(b)

Positive staining (NF-B) (%)

Positive staining (CD68) (%)

50

##

50
40

30

20
10
0

CD

HFD

HFD-N

HFD-S

(d)

Figure 1: Niacin and simvastatin reduced the expressions of CD68 and NF-B p65 in the arterial wall of guinea pigs by immunohistochemistry
analysis after treatment for 8 weeks. (a) and (c) show the representative immunostained aortic sections of CD68 and NF-B p65, respectively
(20x magnification; blue = nuclei and brown = target protein). (b) and (d) show the relative levels of CD68 and NF-B positive cells of per
view field by densitometric quantitation, respectively. Data are presented as mean SD ( = 8). ## < 0.01 versus CD group; < 0.01
versus HFD group.

and 1(b)). These results indicated that niacin weakened the

adhesion and invasion of monocytes in the arterial wall.
NF-B is a transcription factor associated with the expression of various proinflammatory mediators [12]. When not
stimulated, it is found in the cytoplasm connected to its
inhibiting protein, inhibitor B kinase (IB). Stimulation
by inflammatory factors causes degradation of IB protein
and then translocates NF-B to the nucleus. In nucleus, NFB upregulates gene expressions of inflammatory molecules
including chemokines, cytokines, adhesion molecules, and
proteases [13]. In order to further explore the mechanism
through which niacin inhibited inflammatory progress, we
determined the expression of nuclear protein NF-B p65
in the arterial wall by immunohistochemistry analysis and
western blot. The results all indicated that, compared with

CD group, high fat diet promoted the expression of active

NF-B p65 in the arterial wall ( < 0.01); compared with
HFD group, niacin and simvastatin significantly decreased
the expression (Figures 1(c), 1(d), 2(a), and 2(b)).
3.1.3. Niacin Attenuated Oxidative Stress in Guinea Pigs Fed
High Fat Diet. Oxidative stress plays an important role in
the inflammatory process [14]. MDA is one of the most
reliable and widely used indices of oxidative stress [15].
In our study, we determined MDA level in plasma. As shown
in Figure 7, compared with that of CD group, the level of
MDA in plasma was significantly increased in HFD group
( < 0.01). Compared with that of HFD group, niacin and
simvastatin significantly lowered the MDA level by 38% and
43%, respectively (Figure 3).

Mediators of Inflammation
CD

5
HFD

HFD-N

10

HFD-S

##

MDA (nmol/mL) in plasma

NF-Bp65

Histone H3
(a)

1
Relative protein level of nuclear
NF-B in the arterial wall

##
0.8

0.6
0
0.4

0.2
0

CD

HFD

HFD-N

HFD-S

(b)

Figure 2: Niacin and simvastatin suppressed the expression of

nuclear protein NF-B p65 in the arterial wall of guinea pigs fed high
fat diet. The protein expression was analyzed by western blot and
normalized to histone H3 level. (a) shows the representative image
by western blot. (b) shows the IOD ratio of NF-B p65 to Histone
H3. Data are presented as mean SD of at least three independent
experiments. ## < 0.01 versus CD group; < 0.01 versus HFD
group.

3.2. Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflammation in HUVECs

3.2.1. Niacin Attenuated oxLDL-Stimulated Apoptosis of
HUVECs. The inflammation and further damage, including
apoptosis, of arterial ECs are considered as early and necessary steps of AS as well as thrombosis. In this study, HUVECs
apoptosis was analyzed via flow cytometry. After HUVECs
were stained with Annexin V-FITC/PI, flow cytometry analysis revealed that apoptosis was induced by 150 g/mL oxLDL for 24 h, which was significantly improved by niacin
(Figure 4(a)).
3.2.2. Niacin Decreased the Secretion of Inflammatory Cytokines TNF- and IL-6 in oxLDL-Stimulated HUVECs. As
shown in Figures 4(b) and 4(c), ox-LDL (150 g/mL)
markedly increased TNF- and IL-6 protein levels by 76%
and 31%, respectively, in the medium of HUVECs. Preincubation of cells with niacin (0.251 mM) significantly inhibited
TNF- expression by 12, 21, and 27%, respectively. Meanwhile, 1 mM niacin lowered IL-6 level by 15% in the medium.
3.2.3. Niacin Suppressed NF-B p65 and Notch1 Expression in
oxLDL-Stimulated HUVECs. Notch signaling pathway plays
a key role in the inflammatory progress. It can activate
NF-B and promote inflammatory factors secretion from
endothelial cells and macrophages [16]. To further explore
the mechanism through which niacin inhibited cell injury,

CD

HFD

HFD-N

HFD-S

Figure 3: Niacin and simvastatin decreased the level of plasma

MDA in guinea pigs after treatment for 8 weeks. MDA was
determined by a spectrophotometric measurement of thiobarbituric
acid-reactive substances (TBARS) according to the manufacturers
instruction. Data are presented as mean SD ( = 8). ## < 0.01
versus CD group; < 0.01 versus HFD group.

we determined the expressions of nuclear protein NF-B p65

and notch1 by western blot. The results showed that oxLDL
markedly increased the protein levels of active NF-B p65
and notch1 in HUVECs, which were suppressed by preincubation of cells with niacin in a dose-dependent manner
(Figures 4(d), 4(e), 4(f), and 4(g)).
3.3. Niacin Suppressed Inflammatory Response Stimulated by
oxLDL in THP-1 Macrophages
3.3.1. Niacin Decreased TNF- and IL-6 Protein Secretion
in the Medium of THP-1 Macrophages. Next, we assessed
anti-inflammatory property of niacin in THP-1 macrophages.
As shown in Figures 5(a) and 5(b), ox-LDL significantly
promoted TNF- and IL-6 secretion by 89% and 23%,
respectively, in THP-1 macrophages. Niacin (0.251 mM)
remarkably inhibited TNF- expression by 1130% and IL-6
expression by 822% in the medium.
3.3.2. Niacin Inhibited NF-B p65 and Notch1 Protein Expression in oxLDL-Induced THP-1 Macrophages. The effect of
niacin on the protein expressions of NF-B p65 in nuclei and
notch1 stimulated by ox-LDL were examined. Results showed
that niacin (0.251 mM) significantly decreased NF-B level
by 7583% and niacin (1 mM) decreased notch1 level by 20%
(Figures 5(c), 5(d), 5(e), and 5(f)).
3.4. Niacin Significantly Lessened Lipid Deposition in the Arterial Wall and Modified Lipoprotein Profile in Plasma via
Modulating Cholesterol Metabolism in Liver of Guinea Pigs
Fed High Fat Diet
3.4.1. Niacin Significantly Lessened Lipid Deposition in the
Arterial Wall of Guinea Pigs Fed High Fat Diet. Oil red O
staining in the aorta was found in HFD group but not in
CD group because of chow diet without high fat. Compared

Mediators of Inflammation

oxLDL
104

9.61 0.63

103
PI

0 mM niacin

Blank

0.25 mM niacin

25.51 4.62

0.5 mM niacin

18.44 3.40

1 mM niacin

13.71 2.32

14.29 2.82

102
101

7.02 0.85

100
10

10 10 10
Annexin-Y

21.32 1.94
4

10 10

10 10 10
Annexin-Y

14.63 2.68
4

10 10

10
10
10
Annexin-Y

9.02 0.84
4

10 10

10
10
10
Annexin-Y

7.50 1.32
4

10 10

10
102 103
Annexin-Y

104

Relative level of TNF- secretion

##

+
0.25

+
0

+
0.5

+
1

Relative level of IL-6 secretion

(a)

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
oxLDL
Niacin (mM) 0

1.6
##

1.4

1.2
1
0.8
0.6
0.4
0.2

0
oxLDL
Niacin (mM) 0

+
0

+
0.25

(b)

Notch1

Histone H3

-Actin
+
0

+
0.25

+
0.5

+
1

oxLDL
Niacin (mM) 0

+
0

+
0.5

+
1

(e)

##

2.5
2

1.5

1
0.5

0
oxLDL
Niacin (mM) 0

+
0.25

+
0

+
0.25
(f)

+
0.5

+
1

Relative protein level of

notch1 in HUVECs

Relative protein level of

NF-B p65 in nuclei of HUVECs

(d)

3.5
3

+
1

(c)

NF-B p65

oxLDL
Niacin (mM) 0

+
0.5

##
1.5
1

0.5

0
oxLDL
Niacin (mM) 0

+
0

+
0.25

+
0.5

+
1

(g)

Figure 4: Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs. (a) shows the ratio of apoptotic HUVECs stained
with annexin V-FITC and PI. Representative data of flow cytometry analysis are presented. (b) and (c) show the relative levels of TNF- and
IL-6 in the medium of HUVECs. The concentrations of IL-6 and TNF- were determined by ELISA kit. (d) and (e) show the representative
images of NF-B p65 and notch1 protein expression in HUVECs by western blot. (f) and (g) show the IOD ratios of NF-B p65 and notch1
expression, respectively. Data are presented as mean SD. ## < 0.01 versus blank group; < 0.05; < 0.01 versus oxLDL-treated
group without niacin.

Mediators of Inflammation

7
1.4

##

1.5

1
0.5

0
oxLDL
Niacin (mM) 0

+
0

+
0.25

+
0.5

+
1

Relative level of IL-6 secretion

Relative level of TNF- secretion

2.5

##

1.2
1

0.4
0.2

0
oxLDL
Niacin (mM) 0

Notch1

Histone H3

-Actin
+
0

+
0.25

+
0.5

+
1

+
0

+
0.25
(b)

+
0.5

oxLDL
Niacin (mM)

+
0

+
0.25

+
0.5

(c)

(d)

##

Relative protein level of

NF-Bp65 in nuclei

1.2
1
0.8
0.6

0.4

0.2
0
oxLDL
Niacin (mM) 0

+
1

1.4

+
0

+
0.25

+
0.5

+
1

(e)

Relative protein level of notch1

1.4

+
1

0.6

NF-B p65

0.8

(a)

oxLDL
Niacin (mM)

1.2

##

0.8
0.6
0.4
0.2

0
oxLDL
Niacin (mM) 0

+
0

+
0.25

+
0.5

+
1

(f)

Figure 5: Niacin decreased the secretion of TNF- and IL-6 and inhibited NF-B and notch1 expression in oxLDL-stimulated THP-1
macrophages. (a) and (b) show the relative levels of TNF- and IL-6 secretion in the medium of THP-1 macrophages. The concentrations of
IL-6 and TNF- were determined by ELISA kit. (c) and (d) show the representative images of NF-B p65 and notch1 protein expression in
THP-1 macrophages by western blot. (e) and (f) show the IOD ratios of NF-B p65 and notch1 expression, respectively. Data are presented as
mean SD. ## < 0.01 versus blank group; < 0.05; < 0.01 versus oxLDL-treated group without niacin.

with that of HFD group, niacin and simvastatin significantly

decreased the percentages of stained area to the total crosssectional vessel wall by 56% and 67%, respectively (Figure 6).
The effect of simvastatin was superior to that of niacin.
3.4.2. Niacin Increased HDL-C and ApoA I Levels and
Decreased TG and Non-HDL-C Levels in Plasma of Guinea
Pigs Fed High Fat Diet. As shown in Figure 7, after high
fat diet for 8 weeks, the levels of plasma TC, TG, HDL-C,
and non-HDL-C were significantly increased in HFD group
compared with CD group ( < 0.01), which indicated a
successful hyperlipidemic model in guinea pigs. Compared
with HFD group, niacin decreased the levels of TG and

non-HDL-C by 27% and 12%, respectively, and increased

HDL level by 21%. Niacin had no statistical influence on
TC level in plasma. Compared with HFD group, simvastatin
decreased the levels of TG, non-HDL-C, and TC by 18%,
53%, and 51%, respectively. Simvastatin had no significant
influence on HDL-C level.
The level of apoA I in plasma was also detected by SDSPAGE in this study. Compared with that of HFD group, niacin
significantly promoted the level of apoA I by 42%, whereas
simvastatin had no significant influence on apoA I (Figure 8).
3.4.3. Niacin Significantly Upregulated the mRNA Amount
of CYP7A1 in Liver. Cholesterol metabolism in liver is a

Mediators of Inflammation

CD

LDL-R mRNA levels, but simvastatin upregulated LDL-R

mRNA level by 71%.
Cholesterol in liver can be converted into bile acid
through cytochrome P450-meidiated oxidation. The ratelimiting enzyme for the dominant pathway of bile acid
synthesis is cytochrome P450 7A1 (CYP7A1). As shown in
Figure 9(c), compared with HFD group, niacin significantly
upregulated the CYP7A1 mRNA level by 59%, whereas
simvastatin had no significant influence on its level. HMGCR
is the rate-limiting enzyme in the process of cholesterol
synthesis. Compared with that of CD group, the mRNA level
of HMGCR was significantly decreased in HFD group ( <
0.01). Compared with HFD group, simvastatin upregulated
the HMGCR mRNA levels by 46%, whereas niacin had no
significant influence on its level (Figure 9(d)).

HFD

4. Discussion
HFD-N

HFD-S
(a)

Oil red O stained area (%)

##

1.5

0.5

CD

HFD

HFD-N

HFD-S

(b)

Figure 6: Niacin and simvastatin significantly lessened lipid deposition in the arterial wall of guinea pigs fed high fat diet. Lipid
deposition in the aorta wall was analyzed by oil red O staining after
treatment for 8 weeks. The quantification of stained lipids was determined by calculating the percentage of the positive area to the total
cross-sectional vessel wall area by Image-Pro Plus software. Data are
presented as mean SD ( = 8). ## < 0.01 versus CD group;

< 0.01 versus HFD group.

complicated homeostasis involving multiple steps, including

cholesterol ingression, synthesis, and conversion. SR-B1 and
LDL-R in liver play a critical role in cholesterol ingression.
SR-B1 is the HDL receptor on the hepatocyte surface. LDLR can bind to LDL and VLDL and internalize them into
hepatocytes [17]. In the study, we determined the effect of
niacin on the expressions of SR-B1 and LDL-R mRNA in
liver. As shown in Figures 9(a) and 9(b), after treatment with
high fat diet for 8 weeks, the LDL-R mRNA level was downregulated ( < 0.01) and the SR-B1 mRNA level was
not significantly changed in HFD group. Compared with
HFD group, niacin had no significant effect on SR-B1 and

For the first time, to our knowledge, this report demonstrates

niacin inhibited vascular inflammation in guinea pig fed
high fat diet and suppressed oxLDL-stimulated inflammatory
response, even injury, of endothelial cells and macrophages
in vitro. The result indicates a new mechanism for niacins
protective action on cardiovascular disease in addition to its
established effects on lipid metabolism.
The augmentation of inflammatory response has been
clearly documented in pathogenesis of vascular impairment.
The chronic inflammatory pathogenesis in the arterial wall is
as follows. Harmful substances in blood, such as hypercholesterolemia, can induce endothelial dysfunction. This causes
the production of ROS and the secretion of cellular adhesion
molecules (CAMs), cytokines, and chemokines which facilitate adherence and endothelial transmigration of leukocytes
(monocytes and T helper lymphocytes). Monocytes in the
arterial wall will be activated by proinflammatory cytokines
and differentiated into macrophages. Activated macrophages
increase the expression of CAMs and cytokines, which results
in recruitment of more leukocytes into the arterial wall, activates the complement pathways of immune system and the
acute phase response, stimulates proliferation and migration
of smooth muscle cells (SMCs), and promotes fibrous tissue
deposition [18]. In progress, the signaling molecule NF-B
is a proinflammatory major switch that can upregulate the
expression of lots of cytokines [19]. Activated NF-B can
result in the enhanced efflux of TNF- and IL-6 in serum
[20]. Early events in AS are usually driven by NF-B and the
disruption of NF-B signaling pathway has been shown to
slow down the vascular impairment [21].
In the present study we demonstrate that niacin attenuated vascular inflammation induced by high fat diet in vivo.
The involved evidences are as follows. (1) Niacin lowered the
number of macrophages (CD68 positive cells) in the arterial
wall and significantly downregulated the inflammatory factors (IL-6 and TNF-) levels in plasma of guinea pigs fed high
fat diet. (2) Both immunohistochemistry and western blot
analysis indicated niacin suppressed the expression of active
NF-B p65 in nuclei of the arterial wall. The activated NF-B
is reported to form a heterodimer, which usually consists of
two proteins, p65 and p50 subunits. The p65 subunit has been

Mediators of Inflammation

250

2000

##

##

1500

TC (mg/dL)

TG (mg/dL)

200
150
100

1000

500

50
0

CD

HFD

HFD-N

HFD-S

CD

HFD

(a)

HFD-S

(b)

2000

120

100

##

##

Non-HDL-C (mg/dL)

HDL-C (mg/dL)

HFD-N

80
60
40

1500

1000

500

20
0

CD

HFD

HFD-N

HFD-S

HFD

CD

(c)

HFD-N

HFD-S

(d)

Human

160
110
80
60
50
40
30
apoAI

20

Fold induction of apoAI in HDL

HFD-S

HFD-N

HFD

CD

Marker

Figure 7: Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet. The levels of TG (a), TC (b), HDL-C (c), and
non-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeks treatment. Data are presented as mean SD
( = 8). ## < 0.01 versus CD group; < 0.05; < 0.01 versus HFD group.

1.5
#
1

0.5

0
CD
(a)

HFD

HFD-N

HFD-S

(b)

Figure 8: Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet. HDL (density = 1.091.24 g/mL) was separated
from plasma of guinea pigs by sequential ultracentrifugation. The SDS-PAGE was performed on 15% SDS polyacrylamide gel, and the
apolipoproteins were stained with coomassie brilliant blue. Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plus
software. (a) shows the representative SDS-PAGE image of HDL. (b) shows the relative level of apoA I in plasma by densitometric quantitation.
Data are presented as mean SD of at least three independent experiments. # < 0.05 versus CD group; < 0.01 versus HFD group.

10

Mediators of Inflammation
1.2
Relative mRNA level of LDL-R

Relative mRNA level of SR-B1

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

CD

HFD

HFD-N

0.6

##

0.4
0.2
0

HFD-S

0.8

CD

HFD

(a)

(b)

Relative mRNA level of HMGCR

Relative mRNA level of CYP7A1

HFD-S

1.4

1.5

0.5

HFD-N

CD

HFD

HFD-N

HFD-S

(c)

1.2
1
#

0.8
0.6
0.4
0.2
0

CD

HFD

HFD-N

HFD-S

(d)

Figure 9: Effect of niacin and simvastatin on the mRNA abundance of SR-B1, LDL-R, CYP7A1, and HMGCR in liver of guinea pigs fed
high fat diet. The mRNA levels, which were analyzed by quantitative real-time PCR, were calculated after being adjusted for -actin using
the 2Ct method. Data are presented as mean SD of at least three independent experiments. # < 0.05; ## < 0.01 versus CD group;

< 0.01 versus HFD group.

demonstrated to exert critical activity on the transcription

of many inflammatory genes, including adhesion molecules,
cytokines, and chemokines [22]. (3) CRP, an early acute phase
reactant, is closely relevant to inflammation. Baseline level of
CRP is a strong independent predictor of the risk of future
myocardial infarction, peripheral vascular disease, stroke,
and vascular death among healthy individuals without known
vascular disease [23]. Kuvin et al. have shown that niacin
decreased CRP level by 15% in patients with stable coronary
artery disease [5]. In patients with metabolic syndrome,
after treatment with extended-release niacin (1 g/day) for 52
weeks, their endothelial function was improved by 22% and
there was a decrease in CRP level by 20% [24]. Our results
also showed niacin slightly lowered CRP level but had
no statistical difference (Table 1). (4) Oxidative stress was
suppressed by niacin. Oxidative stress is closely related to the
inflammation in the arterial wall. Increased ROS production
can initiate a cascade of signal transduction, which results in
endothelial dysfunction, changes in vascular tone, vascular
remodeling, and vascular inflammatory responses [25].
To further confirm the direct anti-inflammatory property of niacin, its effect on oxLDL-induced inflammatory
response of endothelial cells and macrophages was studied.
oxLDL is pivotal in the development of AS and represents
a crucial proinflammatory stimulus [26]. Upon entering
into the intima of arteries, oxLDL activates endothelial

cells and upregulates adhesion molecule expression and

inflammatory factors secretion, all of which contribute to
the recruitment of circulating leukocytes. Monocytes and/or
macrophages infiltrating the arterial wall take up oxLDL and
form foam cells, which in turn promote further secretion
of inflammatory mediators [27]. Our data indicated niacin
remarkably downregulated the secretion of TNF- and IL-6
stimulated by oxLDL in HUVECs and THP-1 macrophages.
Notch1 signal pathway is an evolutionarily highly conserved
mechanism for communication, which can improve NF-B
activity and have a positive correlation with the development
of inflammation [28]. A previous study demonstrates that
notch knockout in macrophage markedly downregulates the
expression of proinflammatory factors. Instead, when the
notch signal pathway is activated, NF-B expression increases
and inflammatory factors secretion is also promoted [29].
Our findings indicated niacin decreased the expressions of
NF-B and notch1 in oxLDL-induced inflammation. Vascular endothelial apoptosis plays an important role in the
initiation of AS. It may compromise vessel wall permeability
to cytokines, growth factors, lipids, and immune cells and
increase coagulation [30]. In this study, we indicated niacin
attenuated oxLDL-induced apoptosis of HUVECs.
Taken together, these observations lend initial but compelling evidence that niacin has protective effects on cardiovascular disease through its vascular anti-inflammatory

Mediators of Inflammation
properties in addition to its established effects on lipid
metabolism.
Reverse cholesterol transport (RCT) is a potent defense
mechanism against AS. It involves transporting cholesterol
from peripheral tissues and cells to liver, transforming cholesterol into bile acid, and finally eliminating it from the body
[31]. HDL and apoA play a key role in RCT. They can drive
cholesterol efflux from macrophage foam cells that reside
in vessel intima [32, 33]. Similar to previous studies, our
findings also indicated that niacin increased the levels of
HDL and apoA I in plasma significantly. In the process of
RCT, CYP7A1 is an important regulatory factor which can
convert cholesterol to bile acid in liver and be further excreted
into intestine for elimination from body with feces. Here, we
showed that the mRNA abundance of CYP7A1 in liver was
significantly upregulated by niacin treatment. These findings
suggest that niacin may modulate plasma lipid and exert
antiatherosclerotic effects by promoting RCT progress.
In clinical practice, the oral dose of niacin for lipidlowering purpose is usually 13 g per day, equivalently 14
43 mg/kg per day. According to the formula of Meeh-Rubner
equation, we calculate the dose for guinea pig to be about
80250 mg/kg per day. In addition, a recent report showed
that up to 400 mg/kg per day of niacin treatment for 7 days
and 100 mg/kg per day of niacin treatment for 4 weeks are
safe and do not cause toxicity in rodent models [34]. In this
study, the applied dose of niacin for guinea pig is 100 mg/kg/d,
which has pharmacologic effects and does not have various
toxicities based on the changes in food intake, body weight,
and behaviors. In human, plasma level of niacin was found
to be about 0.3 mM after oral ingestion of 1 g niacin [35,
36]. Since the reported plasma concentrations are one time
measurement, it is unclear whether these levels reflect trough
or peak values. Furthermore, there is no reported clinical
data available regarding plasma concentration of niacin after
ingestion of 3 g niacin. Extrapolation of the available data
using oral dose of 1 g niacin would indicate that the 3 g niacin
oral administration may lead to the plasma concentrations of
niacin in the range of 0.81 mM. In our in-vitro studies we
used 0.25 to 1 mM niacin.
Statins are the first-line therapy in the treatment of
cholesterol-induced atherosclerotic cardiovascular diseases
[37]. They suppress the conversion of HMG-CoA to mevalonate by competitively blocking HMGCR; therefore, the
endogenous cholesterol synthesis in the liver is remarkably
reduced. Consequently, it results in the increase of LDL-R
expression on hepatic cell surfaces and the decrease of LDL-C
in plasma [38]. Meanwhile, inhibition of cholesterol synthesis
causes an overexpression of HMGCR due to a feedbackregulatory mechanism [39]. Similar to previous studies, our
findings also revealed that simvastatin significantly decreased
the level of non-HDL-C in plasma followed by upregulating
LDL-R expression in liver and the HMGCR mRNA level in
liver was increased in a compensatory way.

5. Conclusion
In summary, our data presented herein support the novel
concept that niacin has vascular anti-inflammatory and

11
potentially vascular-protective property which is independent of its effect on lipid regulation. The anti-inflammatory
property of niacin is realized by downregulating nuclear
transcription factor-B signaling pathway. Meanwhile, our
finding also suggests that niacin modulates plasma lipid by
stimulating the expression of factors involved in the process
of RCT and promoting RCT.

Conflict of Interests
The authors declare that they have no conflict of interest.

Authors Contribution
Yanhong Si and Ying Zhang have equally contributed to this
study.

Acknowledgments
This research was supported by the financial supports
from the Taishan scholar foundation of Shandong Province
(200867) and the Natural Science Foundations of China
(81170785 and 81371234).

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